The invention is directed to methods for identifying the active domains and amino acid residues in polypeptides. It is also directed to hormone variants.
Polypeptides, i.e., peptides and proteins, comprise a wide variety of biological molecules each having a specific amino acid sequence, structure and function. Most polypeptides interact with specific substances to carry out the function of the polypeptide. Thus, enzymes, such as subtilisin, amylase, tissue plasminogen activator, etc., interact with and hydrolyze specific substrates at particular cleavage sites whereas proteinaceous hormones such as human growth hormone, insulin and the like interact with specific receptors to regulate growth and metabolism. In other cases, the interaction is between the polypeptide and a substance which is not the primary target of the polypeptide such as an immunogenic receptor. Many polypeptides are pluripotential in that they contain discrete regions which interact with different ligands or receptors each of which produces a discrete biological effect. For example, human growth hormone (hGH) is diabetogenic and lypogenic in adults and induces long bone growth in children.
Efforts have been made to modify the primary functional properties of naturally occurring polypeptides by modifying amino acid sequence. One approach has been to substitute one or more amino acids in the amino acid sequence of a polypeptide with a different amino acid. Thus, protein engineering by in vitro mutagenesis and expression of cloned genes reportedly has been applied to improve thermal or oxidative stability of various proteins. Villafranca, J. E., et al. (1983) Science 222, 782-788; Perry, L. J., et al. (1984) Science 226, 555-557; Estell, D. A., et al. (1985) J. Biol. Chem. 260, 6518-6521; Rosenberg, S., et al. (1984) Nature (London) 312, 77-80; Courtney, M., et al. (1985) Nature (London) 313, 149-157. In addition, such methods have reportedly been used to generate enzymes with altered substrate specificities. Estell, D. A., et al. (1986) Science 223, 655-663; Craik, C. S., et al. (1985) Science 228, 291-297; Fersht, A. R., et al. (1985) Nature (London) 314, 235-238; Winther, J. R., et al. (1985) Carlsberg Res. Commun. 50, 273-284; Wells, J. A., et al. (1987) Proc. Natl. Acad. Sci. 84, 1219-1223. The determination of which amino acid residue should be modified has been based primarily on the crystal structure of the polypeptide, the effect of chemical modifications on the function of the polypeptide and/or the interaction of the polypeptide with various substances to ascertain the mode of action of the polypeptide. In some cases, an amino acid substitution has been deduced based on the differences in specific amino acid residues of related polypeptides, e.g. difference in the amino acid sequence in substrate binding regions of subtilisins having different substrate specificities. Wells, J. A., et al. (1987) Proc. Natl. Acad. Sci. USA 84, 5767. In other cases, the amino acid sequence of a known active region of a molecule has reportedly been modified to change that sequence to that of a known active region of a second molecule. Wharton, R. P., et al. (1985) Nature 316, 601-605, and Wharton, R. P., et al. (1984) Cell 38, 361-369 (substitution of recognition helix of phage repressor with recognition helix of different repressor); Jones, P. T., et al. (1986) Nature 321, 522-525 (substitution of variable region of a mouse antibody for corresponding region of human myeloma protein). While this approach may provide some predictability with regard to the properties modified by such substitutions, it is not a methodical procedure which would confirm that all regions and residues determinative of a particular property are identified. At best, empirical estimates of the energetics for the strengths of the molecular contacts of substituted residues may be ascertained. In this manner, the strengths of hydrogen bonds (Fersht, A. R., et al. (1985) Nature 314, 235; Bryan, P., et al. (1986) Proc. Natl. Acad. Sci. USA 83, 3743; Wells, J. A., et al. (1986) Philos. Trans. R. Soc. London A. 317, 415), electrostatic interactions (Wells, J. A., et al. (1987) Proc. Natl. Acad. Sci. USA 84, 1219; Cronin, C. N., et al. (1987) J.Am. Chem. Soc. 109, 2222), and hydrophobic and steric effects (Estell, D. A., et al. (1986) Science 233, 659; Chen, J. T., et al. (1987) Biochemistry 26, 4093) have been estimated for specific modified residues. These and other reports (Laskowski, M., et al. (1987) Cold Spring Harbor Symp. Quant. Biol. 52, 545; Wells, J. A., et al. (1987) Proc. Natl. Acad. Sci. USA 84, 5167; Jones, P. T., et al. (1986) Nature 321, 522; Wharton, R. P., et al. (1985) Nature 316, 601) have concluded that mutagenesis of known contact residues causes large effects on binding whereas mutagenesis of non-contact residues has a relatively minor effect.
A second reported approach to understand the relationship between amino acid sequence and primary function employs in vivo homologous recombination between related genes to produce hybrid DNA sequences encoding hybrid polypeptides. Such hybrid polypeptides have reportedly been obtained by the homologous recombination of trp B and trp A from E.coli and Salmonella typhimurium (Schneider, W. P., et al. (1981) Proc. Natl. Acad. Sci USA 78, 2169-2173); alpha 1 and alpha 2 leukocyte interferons (Weber, H. and Weissmann, C. (1983) Nuc. Acids Res. 11, 5661); the outer membrane pore proteins ompC and phoE from E.coli K-12 (Thommassen, J., et al. (1985) EMBO 4, 1583-1587); and thermophilic alpha-amylases from Bacillus stearothermophilus and Bacillus lichiniformis (Gray, G. L., et al. (1986) J. Bacterial. 166, 635-643). Although such methods may be capable of providing useful information relating to amino acid sequence and function as well as useful hybrid polypeptides, as reported in the case of the hybrid alpha amylases, it is difficult to utilize such methods to systematically study a given polypeptide to determine the precise regions and amino acid residues in the polypeptide that are active with one of the target substances for that particular molecule. This is because the site of crossover recombination, which defines the DNA and amino acid sequence of the hybrid, is determined primarily by the DNA sequence of the genes of interest and the recombination mechanism of the host cell. Such methods do not provide for the predetermined and methodical sequential replacement of relatively small segments of DNA encoding one polypeptide with a corresponding segment from a second gene except in those fortuitous circumstances when crossover occurs near the 5xe2x80x2 or 3xe2x80x2 end of the gene.
The interaction of proteinaceous hormones with their receptors has reportedly been studied by several techniques. One technique uses hormone peptide fragments to map the location of the receptor binding sites on the hormone. The other technique uses competition between neutralizing monoclonal antibodies and peptide fragments to locate the receptor binding site by epitope mapping. Exemplary of these techniques is the work reported on human growth hormone (hGH).
Human growth hormone (hGH) participates in much of the regulation of normal human growth and development. This 22,000 dalton pituitary hormone exhibits a multitude of biological effects including linear growth (somatogenesis), lactation, activation of macrophages, insulin-like effects and diabetagenic effects among others. See Chawla, R. K. (1983) Ann. Rev. Med. 34, 519; Edwards, C. K., et al. (1988) Science 239, 769; Thorner, M. O., et al. (1988) J. Clin. Invest. 81, 745. Growth hormone deficiency in children leads to dwarfism which has been successfully treated for more than a decade by exogenous administration of hGH. There is also interest in the antigenicity of hGH in order to distinguish among genetic and post-translationally modified forms of hGH (Lewis, U. J. (1984) Ann. Rev. Physiol. 46, 33) to characterize any immunological response to hGH when it is administered clinically, and to quantify circulating levels of the hormone.
hGH is a member of a family of homologous hormones that include placental lactogens, prolactins, and other genetic and species variants of growth hormone. Nichol, C. S., et al. (1986) Endocrine Reviews 7, 169. hGH is unusual among these in-that it exhibits broad species specificity and binds monomerically to either the cloned somatogenic (Leung, D. W., et al. (1987) Nature 330, 537) or prolactin receptor (Boutin, J. M., et al. (1988) Cell 53, 69). The cloned gene for hGH has been expressed in a secreted form in Eschericha coli (Chang, C. N., et al. (1987) Gene 55, 189) and its DNA and amino acid sequence has been reported (Goeddel, et al. (1979) Nature 281, 544; Gray, et al. (1985) Gene 39, 247). The three-dimensional structure of hGH is not available. However, the three-dimensional folding pattern for porcine growth hormone (pGH) has been reported at moderate resolution and refinement (Abdel-Meguid, S. S., et al. (1987) Proc. Natl. Acad. Sci. USA 84, 6434).
Peptide fragments from hGH have been used in attempts to map the location of the receptor binding site in hGH. Li, C. H. (1982) Mol. Cell. Biochem. 46, 31; Mills, J. B., et al. (1980) Endocrinology 17, 391. In another report, a fragment consisting of residues 96-133 was isolated after proteolysis of bovine growth hormone. This fragment was reported to bind to a growth hormone receptor. Yamasakin, et al. (1970) Biochemistry 9, 1107. However, when a larger peptide containing residues 1-133 was produced by recombinant methodology, no detectable binding activity was observed. Krivi, G. G., et al., International Symposium on Growth Hormone; Basic and Clinical Aspects, Abstract I-18, Final Program, sponsored by Serono Symposia, USA, Jun. 14-18, 1987. These results are clearly irreconcilable and demonstrate the potential unreliability of using peptide fragments to map receptor binding sites on a proteinaceous hormone, especially for those where the binding site is composed of two or more discontinuous and/or conformationally dependent epitopes.
The use of neutralizing monoclonal antibodies to locate the receptor binding sites by epitope mapping has similar limitations. For example, a monoclonal antibody was reportedly used in a receptor binding assay to compete with the hGH receptor for a peptide consisting of residues 98-128 of hGH. Even though the peptide 98-128 of the hGH hormone only binds to the neutralizing monoclonal antibody, it was proposed that this region contains the receptor binding site based on these competitive studies. Retegin, L. A., et al. (1982) Endocrinology 111, 668.
Similar approaches have been used in attempts to identify antigenic sites on the hGH hormone. Epitope mapping of twenty-seven different monoclonal antibodies to hGH by competitive binding reportedly resolved only four different antigenic sites on the hormone. Surowy, T. K., et al. (1984) Mol. Immunol. 21, 345; Aston, R., et al. (1985) Pharmac. Ther. 27, 403. This strategy, however, did not locate the epitopes on the amino acid sequence of the hormone.
Another approach to defining antigenic sites has been to test the binding of antibodies to short linear peptides over the protein of interest. Geysen, H. M., et al. (1984) Proc. Natl. Acad. Sci. USA 81, 3998; Geysen, H. M. (1985) Immunol. Today 6, 364. However, this approach suffers from the same limitations of using linear peptide fragments to locate receptor binding sites. To be useful, the linear sequence must be capable of adopting the conformation found in the antigen for the antibody to recognize it. Furthermore, based upon the known size of antibody epitopes from X-ray crystallography (Sheriff, S., et al. (1987) Proc. Natl. Acad. Sci USA 84, 8075; Amit, A. G., et al. (1986) Science 23, 747) it has been estimated that virtually all antibody combining sites must be, in part, discontinuous (Barlow, D. J., et al. (1986) Nature 322, 747) and as a result linear fragments may not adequately mimic such structure.
Peptide fragments from hGH have also been studied by non-covalently combining such fragments. Thus, several investigators have reported the analysis of the combination of relatively large fragments of human growth hormone comprising either the natural amino acid sequence or chemically modified peptides thereof. Burstein, S., et al. (1979) J. of Endo. Met. 48, 964 (amino terminal fragment hGH-(1-134) combined with carboxyl-terminal fragment hGH-(141-191)); Li, C. H., et al. (1982) Mol. Cell. Biochem. 46 31; Mills, J. B., et al. (1980) Endocrinology 107, 391 (subtilisin-cleaved two-chain form of hGH).
Similarly, the chemically modified fragment hGH-(1-134) and a chemically modified carboxy-terminal fragment from human chorionic somatomammotropin (also called placental lactogen), (hCS-(141-191)), have been non-covalently combined, as have the chemically modified fragments hCS-(1-133) and hGH-(141-191). U.S. Pat. No. 4,189,426. These investigators reported incorrectly that the determinants for binding to the hepatic growth hormone receptor are in the first 134 amino-terminal residues of growth hormone (Burstein, et al. (1978) Proc. Natl, Acad. Sci. USA 75, 5391-5394). Clearly, such techniques are subject to erroneous results. Moreover, by utilizing two large fragments this technique is only potentially able to localize the function to one or the other of the two fragments used in such combinations without identification of the specific residues or regions actively involved in a particular interaction. A review of some of the above techniques and experiments on hGH has been published. Nichol, C. S., et al. (1986) Endocrine Rev. 7, 169-203.
An alternative approach has been reported wherein a 7 residue peptide fragment from the xe2x80x9cdeletion peptidexe2x80x9d of hGH (hGH-32-46) was modified to contain amino acid residues from analogous segments of growth hormone from other mammalian species. The effect, if any, of such substitutions, however, was not reported. See U.S. Pat. No. 4,699,897. Nonetheless, the shortcomings of the use of short peptide fragments are apparent since the linear sequence of such fragments must be capable of adopting the conformation found in the intact growth hormone so that it may be recognized in an in vitro or in vivo assay.
A number of naturally occurring mutants of hGH have been identified. These include hGH-V (Seeberg, P. H. (1982) DNA 1, 239; U.S. Pat. Nos. 4,446,235, 4,670,393 and 4,665,180) and 20K hGH containing a deletion of residues 32-46 of hGH (Kostyo, J. L., et al. (1987) Biochemica et Biophysica Acta 925, 314; Lewis, U. J., et al. (1978) J. Biol. Chem. 253, 2679).
One investigator has reported the substitution of cysteine at position 165 in hGH with alanine to disrupt the disulfide bond which normally exists between Cys-53 and Cys-165. Tokunaga, T., et al. (1985) Eur. J. Biochem. 153, 445. This single substitution produced a mutant that apparently retained the tertiary structure of hGH and was recognized by receptors for hGH.
Another investigator has reported the in vitro synthesis of hGH on a solid resin support. The first report by this investigator disclosed an incorrect 188 amino acid sequence for hGH. Li, C. H., et al. (1966) J. Am. Chem. Soc. 88, 2050; and U.S. Pat. No. 3,853,832. A second report disclosed a 190 amino acid sequence. U.S. Pat. No. 3,853,833. This latter sequence is also incorrect. In particular, hGH has an additional glutamine after position 68, a glutamic acid rather than glutamine at position 73, an aspartic acid rather than asparagine at position 106 and an asparagine rather than aspartic acid at position 108.
In addition to the foregoizng, hybrid interferons have been reported which have altered binding to a particular monoclonal antibody. Camble, r. et. al. xe2x80x9cProperties of Interferon-xcex12 Analogues Produced from Synthetic Genes in Peptides: Structure and Function,xe2x80x9d Proceedings of the Ninth American Peptide Symposium (1985) eds. Deber et. al., Pierce Chemical Co., Chicago, Ill., pp.375-384. As disclosed therein, amino acid residues 101-114 from xcex1-1 interferon or residues 98-114 from xcex3-interferon were substituted into xcex1-2 interferon. xcex1-2 interferon binds NK-2 monoclonal antibody whereas xcex1-1 interferon does not. This particular region in xcex1-2 interferon apparently was chosen because 7 of the 27 amino acid differences between xcex1-1 and xcex1-2 interferon were located in this region. The hybrids so obtained reportedly had substantially reduced activity with NK-2 monoclonal antibody. When tested for antiviral activity, such hybrids demonstrated antiviral activity on par with the activity of wild type xcex1-2 interferon. Substitutions of smaller sections within these regions were also reported. Sequential substitution of clusters of 3 to 7 alanine residues was also proposed. However, only one analogue [Ala-30,32,33] IFN-xcex12 is disclosed.
Alanine substitutions within a small peptide fragment of hen egg-white lysozyme and the effect of such substitutions on the stimulation of 2A11 or 3A9 cells have also been reported. Allen, P. M., et. al. (1987) Nature 327,713-715.
Others have reported that binding properties can be engineered by replacement of entire units of secondary structure including antigen binding loops (Jones, P. T., et al. (1986) Nature 321, 522-525) or DNA recognition helices (Wharton, R. P., et al. (1985) Nature 316, 601-605).
The references discussed above are provided solely for their disclosure prior to the filing date of the present application, and nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or priority based on earlier filed applications.
Given the state of the art as exemplified by the above references, it is apparent that a need exists for a useful method for the systematic analysis of polypeptides so as to ascertain the relationship between structure and function. Accordingly, it is an object herein to provide such methods to identify the active domains within the polypeptide which contribute to the functional activity of the polypeptide.
It is a further object herein to provide methods for determining the active amino acid residues which determine functional activity.
A further object of the invention is to provide methods for systematically identifying the biologically active domains in a polypeptide.
Further, it is an object herein to provide hormone variants having desirable biological, biochemical and immunogenic properties which are different as compared to the same properties of the hormone from which such variants are derived.
Still further it is an object herein to provide hormone variants having diminished activity with one biological function and substantial or increased activity with a second target substance.
Still further it is an object herein to provide hGH variants having modified binding and/or biological activity with the somatogenic receptor for hGH and increased potency.
Still further it is an object herein to provide hGH variants which retain one or more desirable biological properties but which also have decreased diabetogenic activity.
Further, it is an object herein to provide hPRL and hPL variants having an increased binding activity with the somatogenic receptor of hGH.
Further, it is an object herein to provide DNA sequences, vectors and expression hosts containing such vectors for the cloning and expression of polypeptide variants including hGH variants.
In one aspect, the invention provides methods for the systematic analysis of the structure and function of polypeptides by identifying unknown active domains which influence the activity of the polypeptide with a first target substance. Such unknown active-domains in one aspect of the invention may comprise at least two discontinuous amino acid segments in the primary amino acid sequence of the polypeptide. Active domains are determined by substituting selected amino acid segments of the polypeptide (referred to as the parent polypeptide) with an analogous amino acid segment from an analog to the polypeptide. The analog has a different activity with the target substance as compared to the parent polypeptide. The segment-substituted polypeptides so formed are assayed to determine the activity of each of the segment-substituted polypeptides with the target substance. Such activities are compared to the same activity for the parent polypeptide. Since the structurally analogous amino acid segments are obtained from an analog that has a different interaction with the target substance, a comparison of such activities provides an indication of the location of the active domain in the parent polypeptide.
The method further comprises identifying the active amino acid residues within the active domain of the parent polypeptide. The method comprises substituting a scanning amino acid for one of the amino acid residues within the active domain of the parent polypeptide and assaying the residue-substituted polypeptide so formed with a target substance. The activity of each of the residue-substituted polypeptides is compared to the same activity of the parent polypeptide. These steps are repeated for different amino acids in the active domain until the active amino acid residues are identified.
In another aspect, the invention provides methods to identify different active domains and active amino acid residues for different target substances. Such methods comprise repeating the foregoing methods with a second target.
In accordance with the foregoing method, polypeptide variants are identified which have a different activity with one or more target substance as compared to a parent polypeptide. Such variants are produced based on the identification of the active domains or the identification of the active amino acid residues in the active domain which determine the activity of the parent polypeptide with a target substance.
The invention further comprises growth hormone, prolactin, and placental lactogen variants comprising at least three portions. The first portion corresponds to at least a part of the amino acid sequence of a parent hormone, the third portion corresponds to the amino acid sequence of at least part of the same parent hormone, and the second portion corresponds to an amino acid sequence of an analog to the parent hormone. The second portion is analogous to those amino acid residues of the parent hormone not contained between the first and third portions of the polypeptide variant.
The invention also includes specific human growth hormone,human prolactin and human placental lactogen variants comprising segment-substituted and residue-substituted variants of hGH.